In nature, microorganisms have to live in a variety of environments. Microorganisms are forced to survive under poor nutrition, low temperature, high temperature, and pH change, and particularly in a living body, under the presence of phagocytes or antibacterial humoral factors (such as complements, antibodies, and lysozyme). In such circumstances, bacteria have acquired a mechanism for acutely sensing changes of the growth environments. It has been clarified that, as one of such mechanisms, microorganisms sense their concentrations in the environments via specific signaling substances and cleverly control a variety of their bioactivities depending on the concentrations. Such intercellular signaling mechanism is referred to as a quorum sensing system.
The quorum sensing has been reported for the first time in luminescent marine bacteria, Vibrio fischeri and Vibrio harveyi. However, in recent years, the quorum sensing is recognized as a general gene regulatory mechanism in a variety of bacteria. This phenomenon enables bacteria to concurrently show activities such as bioluminescence, swarming, formation of biofilms, production of proteases, synthesis of antibiotics, development of gene-recipient ability, plasmid conjugational transfer, production of pathogenic factors, and spore formation.
A bacterium having a quorum sensing system synthesizes and releases a signaling molecule, called an autoinducer, and controls gene expression as a function of cell density in response to the signaling molecule. So far, acyl homoserine lactone has been identified as autoinducer-1, and 4,5-dihydroxy-2,3-pentanedione has been identified as autoinducer-2.
It has been reported that clinically important bacteria, such as Vibrio bacteria, Pseudomonas aeruginosa, Serratia and Enterobacter, use autoinducer-1 for the quorum sensing. It has also been reported that Vibrio harveyi uses autoinducer-1, which has high species specificity, for intraspecific communication and uses autoinducer-2, which has low species specificity, for interspecific communication (see, for example, Bassler et al., Bacteriol. 179, pp. 4043-4045, 1997).
Recent studies further show that production of pathogenic factors is regulated by interspecific quorum sensing of pathogenic bacteria using autoinducer-2 (see, for example, Xavier KB. et al., Nature, 437, pp. 750-753, 2005). Therefore, it is required to quantify autoinducer-2 and identify a compound which inhibits the autoinducer-2 activity.
As a method of quantifying autoinducer-2, a bioassay using a bacterium which recognizes an autoinducer followed by emitting light was reported, and a reporter strain of Vibrio harveyi which can emits light in response to only autoinducer-2 was constructed (see, for example, Bassler et al., Mol. Microbiol., 9, pp. 773-786, 1993; and Bassler et al., Mol. Microbiol., 13, pp. 273-286, 1994). To quantify autoinducer-2 by a bioassay using such a bacterium, it is necessary to prepare a calibration curve using a standard sample. However, autoinducer-2 to be measured has an unstable structure and is difficult to obtain, therefore, it is unrealistic to use autoinducer-2 as a standard sample for preparing the calibration curve. Therefore, conventional measurement of autoinducer-2 provides only relative values.
Keersmaecker et al., J. Biol. Chem., 280, 20, pp. 19563-19568, 2008 describes that 4-hydroxy-5-methyl-3(2H)-furanone (hereinafter, also referred to as “HMF”) has an autoinducer-2 activity. However, it is shown that the above-mentioned bioassay, which uses the reporter strain of Vibrio harveyi, can detect the activity at a considerably higher concentration of HMF as compared to 4,5-dihydroxy-2,3-pentanedione (DPD), which is used as autoinducer-2. Therefore, HMF has been considered to be inappropriate as a standard substance for quantifying autoinducer-2.